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Abstract Bacterial spores owe their incredible resistance capacities to molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography on lamellae generated by cryo-focused ion beam micromachining provides insights into the ultrastructural organization of Bacillus subtilis sporangia. The reconstructed tomograms reveal that early during sporulation, the chromosome in the forespore adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a stack of amorphous or structured layers with distinct electron density, dimensions and organization. By analyzing mutant strains using cryo-electron tomography and transmission electron microscopy on resin sections, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of coat morphogenetic proteins.

Pancreatic cancer is associated with a poor prognosis despite multimodal treatments. To improve the efficacy of radiotherapy, the use of nanoscintillators is emerging. Made of high‐Z elements, they absorb X‐rays more efficiently than tissues and can locally enhance the radiation dose provided they have accumulated near tumor cells. This study focuses on the role of the coating, a key parameter that controls both in vitro and in vivo properties of nanoparticles, including their internalization, biocompatibility, and therapeutic efficacy. Polyethylene glycol and tripolyphosphate molecules are used to coat lanthanum fluoride nanoscintillators, and their properties are evaluated on pancreatic cancer models. The experiments demonstrate a higher internalization of the nanoparticles when coated with tripolyphosphate, in both 2D and 3D culture models, correlating with greater efficacy under X‐rays, which may be associated with higher radiation dose‐enhancement. The nanoparticles are also injected intravenously in healthy or tumor‐bearing mice in order to study their toxicity, pharmacokinetics, and biodistribution. Despite a strong liver and spleen accumulation, especially for the tripolyphosphate‐coated nanoparticles, no toxicity is observed for either coating. Because they show promising radiation dose‐enhancement in vitro in both culture models and a limited toxicity in vivo, polyethylene glycol‐coated nanoparticles are good candidates for biomedical applications.

The high capacity of the skeletal muscle to regenerate is due to the presence of muscle stem cells (MuSCs, or satellite cells). The E3 ubiquitin ligase Parkin is a key regulator of mitophagy and is recruited to mitochondria during differentiation of mouse myoblast cell line. However, the function of mitophagy during regeneration has not been investigated in vivo. Here, we have utilized Parkin deficient (Parkin-/-) mice to investigate the role of Parkin in skeletal muscle regeneration. We found a persistent deficiency in skeletal muscle regeneration in Parkin-/- mice after cardiotoxin (CTX) injury with increased area of fibrosis and decreased cross-sectional area (CSA) of myofibres post-injury. There was also a significant modulation of MuSCs differentiation and mitophagic markers, with altered mitochondrial proteins during skeletal muscle regeneration in Parkin-/- mice. Our data suggest that Parkin-mediated mitophagy plays a key role in skeletal muscle regeneration and is necessary for MuSCs differentiation.

Heart failure with preserved ejection fraction (HFpEF) is associated with exercise intolerance due to alterations in the skeletal muscle (SKM). Leucine supplementation is known to alter the anabolic/catabolic balance and to improve mitochondrial function. Thus, we investigated the effect of leucine supplementation in both a primary and a secondary prevention approach on SKM function and factors modulating muscle function in an established HFpEF rat model. Female ZSF1 obese rats were randomized to an untreated, a primary prevention, and a secondary prevention group. For primary prevention, leucine supplementation was started before the onset of HFpEF (8 weeks of age) and for secondary prevention, leucine supplementation was started after the onset of HFpEF (20 weeks of age). SKM function was assessed at an age of 32 weeks, and SKM tissue was collected for the assessment of mitochondrial function and histological and molecular analyses. Leucine supplementation prevented the development of SKM dysfunction whereas it could not reverse it. In the primary prevention group, mitochondrial function improved and higher expressions of mitofilin, Mfn-2, Fis1, and miCK were evident in SKM. The expression of UCP3 was reduced whereas the mitochondrial content and markers for catabolism (MuRF1, MAFBx), muscle cross-sectional area, and SKM mass did not change. Our data show that leucine supplementation prevented the development of skeletal muscle dysfunction in a rat model of HFpEF, which may be mediated by improving mitochondrial function through modulating energy transfer.

Membrane lipid composition is a key factor for bacterial stress adaptation. Here, authors show that ViaA and AAA+ ATPase RavA, alter lipid composition and modulate E. coli aminoglycoside sensitivity.

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome associated with a high morbidity and mortality rate. Leucine supplementation has been demonstrated to attenuate cardiac dysfunction in animal models of cachexia and heart failure with reduced ejection fraction (HFrEF). So far, no data exist on leucine supplementation on cardiac function in HFpEF. Thus, the current study aimed to investigate the effect of leucine supplementation on myocardial function and key signaling pathways in an established HFpEF rat model. Female ZSF1 rats were randomized into three groups: Control (untreated lean rats), HFpEF (untreated obese rats), and HFpEF_Leu (obese rats receiving standard chow enriched with 3% leucine). Leucine supplementation started at 20 weeks of age after an established HFpEF was confirmed in obese rats. In all animals, cardiac function was assessed by echocardiography at baseline and throughout the experiment. At the age of 32 weeks, hemodynamics were measured invasively, and myocardial tissue was collected for assessment of mitochondrial function and for histological and molecular analyses. Leucine had already improved diastolic function after 4 weeks of treatment. This was accompanied by improved hemodynamics and reduced stiffness, as well as by reduced left ventricular fibrosis and hypertrophy. Cardiac mitochondrial respiratory function was improved by leucine without alteration of the cardiac mitochondrial content. Lastly, leucine supplementation suppressed the expression and nuclear localization of HDAC4 and was associated with Protein kinase A activation. Our data show that leucine supplementation improves diastolic function and decreases remodeling processes in a rat model of HFpEF. Beneficial effects were associated with HDAC4/TGF-β1/Collagenase downregulation and indicate a potential use in the treatment of HFpEF.

HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated autocleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization, and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins. Notably, upon drug administration, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted no effect but synergized with CHMP2A-NS3. Localization studies demonstrated the relocalization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

Fungal secretomes are known to contain a multitude of components involved in nutrition, cell growth or biotic interactions. Recently, extra-cellular vesicles have been identified in a few fungal species. Here, we used a multidisciplinary approach to identify and characterize extracellular vesicles produced by the plant necrotroph Botrytis cinerea. Transmission electron microscopy of infectious hyphae and hyphae grown in vitro revealed extracellular vesicles of various sizes and densities. Electron tomography showed the co-existence of ovoid and tubular vesicles and pointed to their release via the fusion of multi-vesicular bodies with the cell plasma membrane. The isolation of these vesicles and exploration of their protein content using mass spectrometry led to the identification of soluble and membrane proteins involved in transport, metabolism, cell wall synthesis and remodeling, proteostasis, oxidoreduction and traffic. Confocal microscopy highlighted the capacity of fluorescently labeled vesicles to target cells of B. cinerea, cells of the fungus Fusarium graminearum, and onion epidermal cells but not yeast cells. In addition, a specific positive effect of these vesicles on the growth of B. cinerea was quantified. Altogether, this study broadens our view on the secretion capacity of B. cinerea and its cell-to-cell communication.

Abstract One major cause of traumatic injury is firearm-related wounds (i.e., ballistic trauma), common in both civilian and military populations, which is increasing in prevalence and has serious long-term health and socioeconomic consequences worldwide. Common primary injuries of ballistic trauma include soft-tissue damage and loss, haemorrhage, bone fracture, and pain. The majority of injuries are of musculoskeletal origin and located in the extremities, such that skeletal muscle offers a major therapeutic target to aid recovery and return to normal daily activities. However, the underlying pathophysiology of skeletal muscle ballistic trauma remains poorly understood, with limited evidence-based treatment options. As such, this review will address the topic of firearm-related skeletal muscle injury and regeneration. We first introduce trauma ballistics and the immediate injury of skeletal muscle, followed by detailed coverage of the underlying biological mechanisms involved in regulating skeletal muscle dysfunction following injury, with a specific focus on the processes of muscle regeneration, muscle wasting and vascular impairments. Finally, we evaluate novel approaches for minimising muscle damage and enhancing muscle regeneration after ballistic trauma, which may have important relevance for primary care in victims of violence.

Phytoplankton account for a large proportion of global primary production and comprise a number of phylogenetically distinct lineages. Here, Uwizeye et al. use FIB-SEM to study ultrastructural plasticity of 7 distinct taxa and describe how subcellular organisation is linked to energy metabolism.

Efficient and suitable animal models directed to skeletal muscle hypertrophy are highly needed; nevertheless, the currently available models have limitations, such as restricted hypertrophy outcome and prolonged protocols; thus, additional research is required. In this study, we developed an improved muscle training protocol for mice by directly stimulating the tibialis anterior (TA) muscle motor point using electrical stimulation. C57BL/6 adult male mice were separated into four groups: CTR (control groups for one and two weeks), ES1 (electrical stimulation for one week), and ES2 (electrical stimulation for two weeks). Following muscle training, TA was taken for further examination. The results demonstrated a steady increase in the fiber cross-sectional area as a result of muscle training (ES1, 14.6% and ES2, 28.9%, p < 0.0001). Two weeks of muscle training enhanced muscle mass and maximal tetanic force by 18 (p = 0.0205) and 30%, respectively (p = 0.0260). To assess the tissue remodeling response in this model, we evaluated satellite cell activity and observed an increase in the number of Pax-7-positive nuclei after one and two weeks of muscle training (both >2-fold, p < 0.0001). In addition, we observed an increase in the number of positive nuclei for MyoD after two weeks (2.6-fold, p = 0.0057) without fiber damage. Accordingly, phosphorylation of mTOR and p70 increased following two weeks of muscle training (17%, p = 0.0215 and 66%, p = 0.0364, respectively). The results indicate that this muscle training strategy is appropriate for promoting quick and intense hypertrophy.

Synthetic amorphous silica (SAS) is a nanomaterial used in a wide variety of applications, including the use as a food additive. Two types of SAS are commonly employed as a powder additive, precipitated silica and fumed silica. Numerous studies have investigated the effects of synthetic amorphous silica on mammalian cells. However, most of them have used an exposure scheme based on a single dose of SAS. In this study, we have used instead a repeated 10-day exposure scheme in an effort to better simulate the occupational exposure encountered in daily life by consumers and workers. As a biological model, we have used the murine macrophage cell line J774A.1, as macrophages are very important innate immune cells in the response to particulate materials. In order to obtain a better appraisal of the macrophage responses to this repeated exposure to SAS, we have used proteomics as a wide-scale approach. Furthermore, some of the biological pathways detected as modulated by the exposure to SAS by the proteomic experiments have been validated through targeted experiments. Overall, proteomics showed that precipitated SAS induced a more important macrophage response than fumed SAS at equal dose. Nevertheless, validation experiments showed that most of the responses detected by proteomics are indeed adaptive, as the cellular homeostasis appeared to be maintained at the end of the exposure. For example, the intracellular glutathione levels or the mitochondrial transmembrane potential at the end of the 10 days exposure were similar for SAS-exposed cells and for unexposed cells. Similarly, no gross lysosomal damage was observed after repeated exposure to SAS. Nevertheless, important functions of macrophages such as phagocytosis, TNFα, and interleukin-6 secretion were up-modulated after exposure, as was the expression of important membrane proteins such as the scavenger receptors, MHC-II, or the MAC-1 receptor. These results suggest that repeated exposure to low doses of SAS slightly modulates the immune functions of macrophages, which may alter the homeostasis of the immune system.

The bacterial cell wall is in part composed of the peptidoglycan (PG) layer that maintains the cell shape and sustains the basic cellular processes of growth and division. The cell wall of Gram-positive bacteria also carries teichoic acids (TAs). In this work, we investigated how TAs contribute to the structuration of the PG network through the modulation of PG hydrolytic enzymes in the context of the Gram-positive Streptococcus pneumoniae bacterium. Pneumococcal TAs are decorated by phosphorylcholine residues which serve as anchors for the Choline-Binding Proteins, some of them acting as PG hydrolases, like the major autolysin LytA. Their binding is non covalent and reversible, a property that allows easy manipulation of the system.In this work, we show that the release of LytA occurs independently from its amidase activity. Furthermore, LytA fused to GFP was expressed in pneumococcal cells and showed different localization patterns according to the growth phase. Importantly, we demonstrate that TAs modulate the enzymatic activity of LytA since a low level of TAs present at the cell surface triggers LytA sensitivity in growing pneumococcal cells. We previously developed a method to label nascent TAs in live cells revealing that the insertion of TAs into the cell wall occurs at the mid-cell. In conclusion, we demonstrate that nascent TAs inserted in the cell wall at the division site are the specific receptors of LytA, tuning in this way the positioning of LytA at the appropriate place at the cell surface. Keywords: Streptococcus pneumoniae cell wall, Peptidoglycan hydrolases, Choline-Binding Proteins, Major autolysin LytA localization, Teichoic acids localization

Phytoplankton and plant plastids have distinct evolutionary origins and membrane organization. Here Floriet al. show that diatom photosynthetic complexes spatially segregate into interconnected subdomains within loose thylakoid stacks enabling fast diffusion of electron carriers and efficient photosynthesis

In this study we surveyed a rat skeletal muscle RNA-Seq for genes that are induced by hindlimb immobilization and, in turn, become attenuated by leucine supplementation. This approach, in search of leucine-atrophy protection mediating genes, identified histone deacetylase 4 (HDAC4) as highly responsive to both hindlimb immobilization and leucine supplementation. We then examined the impact of leucine on HDAC4 expression, tissue localization, and target genes. A total of 76 male Wistar rats (~280 g) were submitted to hindlimb immobilization and/or leucine supplementation for 3, 7 and 12 days. These animals were euthanized, and soleus muscle was removed for further analysis. RNA-Seq analysis of hindlimb immobilized rats indicated a sharp induction (log2 = 3.4) of HDAC4 expression which was attenuated by leucine supplementation (~50%). Real-time PCR and protein expression analysis by Western blot confirmed increased HDAC4 mRNA after 7 days of hindlimb immobilization and mitigation of induction by leucine supplementation. Regarding the HDAC4 localization, the proportion of positive nuclei was higher in the immobilized group and decreased after leucine supplementation. Also, we found a marked decrease of myogenin and MAFbx-atrogin-1 mRNA levels upon leucine supplementation, while CAMKII and DACH2 mRNA levels were increased by leucine supplementation. Our data suggest that HDAC4 inhibition might be involved in the anti-atrophic effects of leucine.

Patients with malignant tumors frequently suffer during disease progression from a syndrome referred to as cancer cachexia (CaCax): CaCax includes skeletal muscle atrophy and weakness, loss of bodyweight, and fat tissues. Currently, there are no FDA (Food and Drug Administration) approved treatments available for CaCax. Here, we studied skeletal muscle atrophy and dysfunction in a murine CaCax model by injecting B16F10 melanoma cells into mouse thighs and followed mice during melanoma outgrowth. Skeletal muscles developed progressive weakness as detected by wire hang tests (WHTs) during days 13–23. Individual muscles analyzed at day 24 had atrophy, mitochondrial dysfunction, augmented metabolic reactive oxygen species (ROS) stress, and a catabolically activated ubiquitin proteasome system (UPS), including upregulated MuRF1. Accordingly, we tested as an experimental intervention of recently identified small molecules, Myomed-205 and -946, that inhibit MuRF1 activity and MuRF1/MuRF2 expression. Results indicate that MuRF1 inhibitor fed attenuated induction of MuRF1 in tumor stressed muscles. In addition, the compounds augmented muscle performance in WHTs and attenuated muscle weight loss. Myomed-205 and -946 also rescued citrate synthase and complex-1 activities in tumor-stressed muscles, possibly suggesting that mitochondrial-metabolic and muscle wasting effects in this CaCax model are mechanistically connected. Inhibition of MuRF1 during tumor cachexia may represent a suitable strategy to attenuate skeletal muscle atrophy and dysfunction.

Iron oxide nanoparticles/microparticles are widely present in a variety of environments, e.g., as a byproduct of steel and iron degradation, as, for example, in railway brakes (e.g., metro station) or in welding fumes. As all particulate material, these metallic nanoparticles are taken up by macrophages, a cell type playing a key role in the innate immune response, including pathogen removal phagocytosis, secretion of free radical species such as nitric oxide or by controlling inflammation via cytokine release. In this paper, we evaluated how macrophages functions were altered by two iron based particles of different size (100 nm and 20 nm). We showed that at high, but subtoxic concentrations (1 mg/mL, large nanoparticles induced stronger perturbations in macrophages functions such as phagocytic capacity (tested with fluorescent latex microspheres) and the ability to respond to bacterial endotoxin lipopolysaccharide stimulus (LPS) in secreting nitric oxide and pro-cytokines (e.g., Interleukin-6 (IL-6) and Tumor Necrosis Factor (TNF)). These stronger effects may correlate with an observed stronger uptake of iron for the larger nanoparticles.